Prostate cancer is the fastest growing neoplasm in men with an estimated 244,000 new cases in the United States being diagnosed in 1995, of which approximately 44,000 deaths will result. Prostate cancer is now the most frequently diagnosed cancer in men. Prostate cancer is latent; many men carry prostate cancer cells without overt signs of disease. It is associated with a high morbidity. Cancer metastasis to bone (late stage) is common and is almost always fatal.
Current treatments include radical prostatectomy, radiation therapy, hormonal ablation and chemotherapy. Unfortunately, in approximately 80% of cases, diagnosis of prostate cancer is established when the disease has already metastasized to the bones, thus limiting the effectiveness of surgical treatments. A variety of agents are available which are used in androgen blockade therapy and include luteinizing hormone releasing hormone analogs, steroidal anti-androgens such as cyproterone acetate, nonsteroidal anti-androgens such as flutamide, and other agents such as aminoglutethimide and ketoconazole. However, hormonal therapy frequently fails with time with the development of hormone-resistant tumor cells. Although chemotherapeutic agents have been used in the treatment of prostate cancer, no single agent has demonstrated superiority over its counterparts, and no drug combination seems particularly effective. The generally drug-resistant, slow-growing nature of most prostate cancers makes them particularly unresponsive to standard chemotherapy.
A major, indeed the overwhelming, obstacle to cancer therapy is the problem of selectivity; that is, the ability to inhibit the multiplication of tumor cells, while leaving unaffected the function of normal cells. The therapeutic ratio, or ratio of tumor cell killing to normal cell killing of traditional tumor chemotherapy, is only 1.5:1. Thus, more effective treatment methods and pharmaceutical compositions for therapy and prophylaxis of prostatic hyperplasia and neoplasia are needed.
Of particular interest is development of more specific, targeted forms of therapy for prostate diseases. In contrast to conventional cancer therapies, which result in relatively non-specific and often serious toxicity or impotence, more specific treatment modalities attempt to inhibit or kill malignant cells selectively while leaving healthy cells intact.
One possible treatment approach for prostate diseases is gene therapy, whereby a gene of interest is introduced into the malignant cell. Boulikas (1997) Anticancer Res. 17:1471-1505. The gene of interest may encode a protein which converts into a toxic substance upon treatment with another compound, or an enzyme that converts a prodrug to an active drug. For example, introduction of the herpes simplex gene encoding thymidine kinase (HSV-tk) renders cells conditionally sensitive to ganciclovir (GCV). Zjilstra et al. (1989) Nature 342: 435; Mansour et al. (1988) Nature 336: 348; Johnson et al. (1989) Science 245: 1234; Adair et al. (1989) Proc. Natl. Acad. Sci. USA 86: 4574; Capecchi (1989) Science 244: 1288. Alternatively, the gene of interest may encode a compound that is directly toxic, such as diphtheria toxin (DT). For these treatments to be rendered specific to prostate cells, the gene of interest can be under control of a transcriptional regulatory element that specifically (i.e. preferentially) increases transcription of an operably linked polynucleotide in the prostate cells. Cell- or tissue-specific expression can be achieved by using cell-specific enhancers and/or promoters. See generally, Huber et al. (1995) Adv. Drug Delivery Rev. 17:279-292.
A variety of viral and non-viral (e.g., liposomes) vehicles, or vectors, have been developed to transfer these genes. Of the viruses, retroviruses, herpes virus, adeno-associated virus, Sindbis virus, poxvirus and adenoviruses have been proposed for use in gene transfer, with retrovirus vectors or adenovirus vectors being the focus of much current research. Verma and Somia (1997) Nature 389:239-242.
Adenoviruses are among the most easily produced and purified, and furthermore do not integrate into the host genome, reducing the possibility of dangerous mutations. Moreover, adenovirus has the advantage of effecting high efficiency of transduction and does not require cell proliferation for efficient transduction of cell. For general background references regarding adenovirus and development of adenoviral vector systems, see Graham et al. (1973) Virology 52:456-467; Takiff et al. (1981) Lancet 11:832-834; Berkner et al. (1983) Nucleic Acid Research 11: 6003-6020; Graham (1984) EMBO J 3:2917-2922; Bett et al. (1993) J. Virology 67:5911-5921; and Bett et al. (1994) Proc. Natl. Acad. Sci. USA 91:8802-8806.
When used as gene transfer vehicles, adenovirus vectors are often designed to be replication-defective and are thus deliberately engineered to fail to replicate in the target cells of interest. In these vehicles, the early adenovirus gene products E1A and/or E1B are deleted and provided in trans by the packaging cell line 293. Graham et al. (1987) J. Gen. Virol 36:59-72; Graham (1977) J. Genetic Virology 68:937-940. The gene to be transduced is commonly inserted into adenovirus in the deleted E1A and E1B region of the virus genome. Bett et al. (1994). Replication-defective adenovirus vectors as vehicles for efficient transduction of genes have been described by, inter alia, Stratford-Perricaudet (1990) Human Gene Therapy 1:241-256; Rosenfeld (1991) Science 252:431-434; Wang et al. (1991) Adv. Exp. Med. Biol. 309:61-66; Jaffe et al. (1992) Nat. Genet. 1:372-378; Quantin et al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584; Rosenfeld et al. (1992) Cell 68:143-155; Stratford-Perricaudet et al. (1992) J. Clin. Invest. 90:626-630; Le Gal Le Salle et al. (1993) Science 259:988-990 Mastrangeli et al. (1993) J. Clin. Invest. 91:225-234; Ragot et al. (1993) Nature 361:647-650; Hayaski et al. (1994) J. Biol. Chem. 269:23872-23875; and Bett et al. (1994).
The virtually exclusive focus in the development of adenoviral vectors for gene therapy is use of adenovirus merely as a vehicle for introducing the gene of interest, not as an effector in itself. Replication of adenovirus has been viewed as an undesirable result, largely due to the host immune response. In the treatment of cancer by replication-defective adenoviruses, the host immune response limits the duration of repeat doses at two levels. First, the capsid proteins of the adenovirus delivery vehicle itself are immunogenic. Second, viral late genes are frequently expressed in transduced cells, eliciting cellular immunity. Thus, the ability to administer repeatedly cytokines, tumor suppressor genes, ribozymes, suicide genes, or genes which convert a prodrug to an active drug has been limited by the immunogenicity of both the gene transfer vehicle and the viral gene products of the transfer vehicle as well as the transient nature of gene expression. There is a need for vector constructs that are capable of eliminating cancerous cells in a minimum number of administrations before specific immunological response against the vector prevents further treatment.
Human Glandular Kallikrein
Prostate-specific antigen (PSA or hKLK3) and human pancreatic/renal kallikrein are two members of a subgroup of serine proteases that are potentially involved in the activation of specific polypeptides throughout post-translational processing. Clements (1989) Endocr. Rev. 10:393-419. PSA is synthesized exclusively by normal, hyperplastic, and malignant prostatic epithelia; hence, its tissue-specific expression has made it an excellent biomarker for benign prostatic hyperplasia (BPH) and prostatic carcinoma (CaP). Normal serum levels of PSA are typically below 5 ng/ml, with elevated levels indicative of BPH or CaP.
A third member of the kallikrein gene family, human glandular kallikrein-1 (hGK-1 or hKLK2, encoding the hK2 protein), shares a number of characteristics with PSA. First, both are expressed exclusively in the prostate and are up-regulated by androgens primarily by transcriptional activation. Wolf et al. (1992) Molec. Endocrinol. 6:753-762. Morris (1989) Clin. Exp. Pharm. Physiol. 16:345-351; Qui et al. (1990) J. Urol. 144:1550-1556; Young et al. (1992) Biochem. 31:818-824. Second, hKLK2 and PSA mRNAs are synthesized and co-localize only in prostatic epithelia. Third, hK2 and PSA exhibit a high degree of amino acid sequence identity. Schedlich et al. (1987) DNA 6:429-437. Fourth, they have similar regulatory elements. There is approximately 80% nucleotide sequence identity between PSA and hKLK2 in the 5′-flanking region from −300 to −1 relative to the transcription initiation site. Young et al. (1992) Biochem. 31:818-824. Each promoter contains an androgen responsive element (ARE); their respective ARE's differ from one another by only 1 nucleotide. Schedlich et al. (1987) DNA 6:429-437; Murtha et al. (1993) Biochem. 32:6459-6464.
The levels of hK2 found in various tumors and in the serum of patients with prostate cancer differ substantially from those of PSA. Circulating hK2 in different relative proportions to PSA has been detected in the serum of patients with prostate cancer. Charlesworth et al. (1997) Urology 49:487-493. Expression of hK2 has been detected in each of 257 radical prostatectomy specimens analyzed. Darson et al. (1997) Urology 49:857-862. The intensity and extent of hK2 expression, detected using specific antibodies, increased from benign epithelium to high-grade prostatic intraepithelial neoplasia (PIN) and adenocarcinoma, whereas PSA and prostate acid phosphatase (PAP) displayed an inverse pattern of immunoreactivity. Darson et al. (1997) Urology 49:857-862. Indeed, it has been reported that a certain percentage of PSA-negative tumors have detectable hK2. Tremblay et al. (1997) Am. J. Pathol. 150:455-459.
The hKLK2 promoter is inducible by androgen, consistent with the presence in the promoter of an ARE. Murtha et al. (1993). However, the promoter region of approximately 627 base pairs of the 5′ flanking region of the hKLK2 gene, which was linked to a reporter gene in a plasmid construct and introduced into cells, responded with only an approximately 10-fold increase in reporter gene activity when androgen was added to the culture medium. Murtha et al. (1993).
Androgen induction of gene expression requires the presence of an androgen receptor (AR). Typically, an androgen diffuses passively into the cell where it binds AR. The androgen-activated AR binds to specific DNA sequences called androgen-responsive elements (AREs or ARE sites). Once anchored to an ARE, the AR is able to regulate transcriptional activity in either a positive or negative fashion. Lindzey et al. (1994) Vitamins and Hormones 49: 383-432.
The AR belongs to a nuclear receptor superfamily whose members are believed to function primarily as transcription factors that regulate gene activity through binding to specific DNA sequences, hormone-responsive elements. Carson-Jurica et al. (1990) Endocr. Rev. 11: 201-220. This family includes the other steroid hormone receptors as well as the thyroid hormone, the retinoic acid and the vitamin D3 receptors. The progesterone and glucocorticoid receptor are structurally most closely related to the AR. Tilley et al. (1989) Proc. Natl. Acad. Sci. USA 86: 327-331; Zhou et al. (1994) Recent Prog. Horm. Res. 49: 249-274; and Lindzey et al. (1994) Vitamins and Hormones 49: 383-432.
The AR gene itself is a target of androgenic regulation. In the prostate cancer cells lines PC3 and DU145, which do not express an endogenous AR, androgenic up-regulation of AR cDNA expression occurred in the transfected cells. Dai et al. (1996) Steroids 61:531-539. Androgenic up-regulation of AR mRNA and protein was observed in PC3 cells that were stably transfected with the AR cDNA, suggesting that AR mRNA regulation also occurs when the cDNA is organized into chromatin. Dai et al. (1996).
Identification of prostate-specific genes and the transcription regulatory elements that control their expression would facilitate the development of strategies to combat prostate cancer by providing targets for therapy. The development of novel therapeutic approaches to the treatment of prostate cancer is critical, since these diseases are generally recalcitrant to conventional therapies.